Summary We leveraged IDH wild-type glioblastomas, derivative neurospheres, and single cell gene expression profiles to define three tumor-intrinsic transcriptional subtypes designated as proneural, mesenchymal, and classical. Transcriptomic subtype multiplicity correlated with increased intratumoral heterogeneity and presence of tumor microenvironment. In silico cell sorting identified macrophages/microglia, CD4+ T lymphocytes, and neutrophils in the glioma microenvironment. NF1 deficiency resulted in increased tumor-associated macrophages/microglia infiltration. Longitudinal transcriptome analysis showed that expression subtype is retained in 55% of cases. Gene signature-based tumor microenvironment inference revealed a decrease in invading monocytes and a subtype-dependent increase in macrophages/microglia cells upon disease recurrence. Hypermutation at diagnosis or at recurrence associated with CD8+ T cell enrichment. Frequency of M2 macrophages detection associated with short-term relapse after radiation therapy.

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Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors to chemo-and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to cradiation, has the capacity to repopulate the tumors and contribute to their malignant progression. c-radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by c-radiation in CD1331 GSCs. Irradiation of CD1331 cells induced autophagy within 24-48 hr and slightly decreased the viability of the cells. c-radiation induced a larger degree of autophagy in the CD1331 cells as compared with CD1332 cells and the CD1331 cells expressed higher levels of the autophagy-related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD1331 cells to c-radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD1331 GSCs to c-radiation. ' UICCKey words: autophagy; glioma stem cells; c-radiation; ATG5; ATG12 Glioblastomas (GBMs), the most frequent and aggressive primary brain tumors, are characterized by increased proliferation, resistance to chemotherapy and radiotherapy and invasion into the surrounding normal brain tissue. 1,2 Current treatments include surgery, radiation therapy and chemotherapy. 3,4 Unfortunately, the prognosis of patients with GBMs remains extremely poor and has not changed significantly during the past several years. 5,6 Therefore, novel therapeutic approaches are needed to improve the poor prognosis of these patients.Recently, a small subpopulation of CD1331 cancer stem cells has been identified in specimens of GBM. 7,8 These glioma stem cells (GSCs) express additional stem cell markers, exhibit selfrenewal and differentiation to glial and neuronal lineages, and can initiate xenograft tumors. 9,10 Cancer stem cells in various tumors, including the GSCs, have been implicated in the enhanced radioresistance and in the repopulation of tumors following these treatments. 10,11 Thus, delineating the molecular mechanisms underlying the increased resistance of these cells to anticancer therapies is of utmost importance.Autophagy is a cellular pathway involved in protein and organelle degradation. 12,13 This process is regulated by a series of autophagy-related genes (ATGs) and a number of signaling molecules such as mTOR, AKT, and class I and class III phosphatidylinositol 3-kinase. 14,15 Autophagy is frequently activated in tumor cells following anticancer therapies such as chemotherapeutic drugs 16,17 or g-irradiation 1...

To understand how genomic heterogeneity of glioblastoma contributes to the poor response to therapy characteristic of this disease, we performed DNA and RNA sequencing on GBM tumor samples and the neurospheres and orthotopic xenograft models derived from them. We used the resulting data set to show that somatic driver alterations including single nucleotide variants, focal DNA alterations, and oncogene amplification on extrachromosomal DNA (ecDNA) elements were in majority propagated from tumor to model systems. In several instances, ecDNAs and chromosomal alterations demonstrated divergent inheritance patterns and clonal selection dynamics during cell culture and xenografting. We infer that ecDNA inherited unevenly between offspring cells, a characteristic that affects the oncogenic potential of cells with more or fewer ecDNAs. Longitudinal patient tumor profiling found that oncogenic ecDNAs are frequently retained throughout the course of disease. Our analysis shows that extrachromosomal elements allow rapid increase of genomic heterogeneity during glioblastoma evolution, independent of chromosomal DNA alterations.

Glioblastomas (GBM), the most common and aggressive malignant astrocytic tumors, contain a small subpopulation of cancer stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Here, we study the expression and function of miR-137, a putative suppressor miRNA, in GBM and GSCs. We found that the expression of miR-137 was significantly lower in GBM and GSCs compared to normal brains and neural stem cells (NSCs) and that the miR-137 promoter was hypermethylated in the GBM specimens. The expression of miR-137 was increased in differentiated NSCs and GSCs and overexpression of miR-137 promoted the neural differentiation of both cell types. Moreover, pre-miR-137 significantly decreased the self-renewal of GSCs and the stem cell markers Oct4, Nanog, Sox2 and Shh. We identified RTVP-1 as a novel target of miR-137 in GSCs; transfection of the cells with miR-137 decreased the expression of RTVP-1 and the luciferase activity of RTVP-1 3'-UTR reporter plasmid. Furthermore, overexpression of RTVP-1 plasmid lacking its 3'-UTR abrogated the inhibitory effect of miR-137 on the self-renewal of GSCs. Silencing of RTVP-1 decreased the self-renewal of GSCs and the expression of CXCR4 and overexpression of CXCR4 abrogated the inhibitory effect of RTVP-1 silencing on GSC self-renewal. These results demonstrate that miR-137 is downregulated in GBM probably due to promoter hypermethylation. miR-137 inhibits GSC self-renewal and promotes their differentiation by targeting RTVP-1 which downregulates CXCR4. Thus, miR-137 and RTVP-1 are attractive therapeutic targets for the eradication of GSCs and for the treatment of GBM.